Identification of G6PD inhibitors for the development of novel antimalarial drugs

描述(申请人提供)：热带疟疾是由恶性疟原虫引起的，每年造成多达300万人死亡。由于寄生虫对大多数临床可用药物产生抗药性，迫切需要新的抗疟疾药物。葡萄糖-6-磷酸脱氢酶(G6PD)是一种新的抗疟疾药物设计靶点，基于对该酶基因缺陷的观察，该酶可预防疟疾。G6PD催化磷酸戊糖途径的第一步，产生NADPH，这是一种基本的还原，相当于解毒红细胞(RBCs)中的氧化应激。疟疾寄生虫在红细胞阶段容易受到氧化应激的影响。自然产生的G6PD缺乏症导致缺乏还原当量，氧化应激增加，寄生虫感染的红细胞吞噬功能增强，结果是对疟疾的保护。恶性疟原虫感染红细胞中的NADPH不仅由人G6PD产生，而且由一种具有G6PD活性的寄生虫酶产生，称为恶性疟原虫葡萄糖-6-磷酸脱氢酶-6-磷酸葡萄糖内酯酶(PfGluPho)。我们假设，抑制PfGluPho，并在一定程度上抑制人类G6PD，可以降低患疟疾的风险。到目前为止，G6PD和PfGluPho作为抗疟疾药物靶点的探索受到缺乏重组PfGluPho的限制。最近，我们的德国团队生产出了第一个完整的、具有功能的重组PfGluPho。我们现在的目标是使用这种重组的纯蛋白质来识别可能成为新型和创新抗疟疾药物的潜在候选药物的抑制剂。在目标1中，我们将以镁为单位生产人G6PD和PfGluPho。然后，AIM 2在已经建立的高通量筛选试验中使用这些蛋白质来识别酶抑制剂。活性在低微摩尔到纳摩尔浓度范围内的化合物将受到分离酶的详细动力学分析。目的3跟踪已确定的抑制物，并评估它们是否在不对哺乳动物细胞产生细胞毒性的情况下影响恶性疟原虫的生长和寄生虫血症。由于G6PD缺乏症是一种被证实的抗疟疾寄生虫的原理，而且寄生虫酶在结构和机械上与人类宿主酶不同，PfGluPho是一个很好的药物靶点。我们的目标是确定2-3个先导化合物，这些化合物的活性在纳摩尔范围内，没有明显的细胞毒性，可以用于进一步的药物开发。这可能是一种高风险的方法，因为不能保证高通量筛查和后续分析能确定是否有感染。然而，我们已经确定了164种抑制细菌G6PD的化合物，我们预计对PfGluPho和人类G6PD的命中率相似。这种方法有可能产生高影响力的结果。疟疾是世界上最致命的疾病。疟疾寄生虫对目前可用的大多数药物产生抗药性。因此，迫切需要新的和创新的抗疟疾药物。与公共卫生相关：热带疟疾每年造成多达300万人死亡。疟疾寄生虫恶性疟原虫对大多数临床可用药物产生抗药性。迫切需要新的抗疟疾药物。葡萄糖-6-磷酸脱氢酶是抗疟疾药物设计的新靶点，其基础是观察到该酶缺陷的人可以预防疟疾。我们的目标是在疟疾寄生虫中以及在一定程度上抑制人类体内抑制这种酶的化合物。我们的结果可能为新的抗疟疾药物的开发铺平道路。

DESCRIPTION (provided by applicant): Tropical malaria, caused by the parasite Plasmodium falciparum, is responsible for up to three million deaths each year. Since the parasite develops resistance against most clinically available drugs, novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase (G6PD) is a novel target for antimalarial drug design based on observations that humans with a genetic deficiency in this enzyme are protected against malaria. G6PD catalyses the initial step of the pentose phosphate pathway, yielding NADPH, an essential reducing equivalent to detoxify oxidative stress in red blood cells (RBCs). The malaria parasite is susceptible to oxidative stress in the RBC stage. Naturally occurring G6PD deficiency leads to a lack of reducing equivalents, an increase in oxidative stress, enhanced phagocytosis of parasite-infected RBCs, and, as a consequence, to a protection against malaria. NADPH in parasite-infected RBCs is generated by human G6PD but also by a parasite enzyme with G6PD activity, called P. falciparum glucose-6-phosphate-dehydrogenase-6- phosphogluconolactonase (PfGluPho). We hypothesize that inhibiting PfGluPho and, to a certain extent, human G6PD reduces the risk of developing malaria. So far, exploring G6PD and PfGluPho as antimalarial drug targets was limited by a lack of recombinant PfGluPho. Very recently our German team produced the first complete and functional recombinant PfGluPho. We now aim at using this recombinant, pure protein to identify inhibitors which could be potential candidates for novel and innovative antimalarial drugs. In AIM 1 we will produce human G6PD and PfGluPho in mg quantities. AIM 2 then uses these proteins in already established high-throughput screening assays to identify enzyme inhibitors. Compounds active in the low micromolar to nanomolar concentration range will be subject to detailed kinetic analyses on isolated enzymes. AIM 3 follows up on the identified inhibitors and assesses whether they impact P. falciparum growth and parasitemia without being cytotoxic in mammalian cells. Since G6PD deficiency is a proven principle against malarial parasites and since the parasite enzyme differs structurally and mechanistically from the human host enzyme, PfGluPho is an excellent drug target. We aim at identifying 2-3 lead compounds which are active in the nanomolar range without significant cytotoxicity, which can be used for further drug development. This may be a high risk approach since it is not guaranteed that high-throughput screening and follow-up assays identify a hit. However, we have already identified 164 compounds that inhibit bacterial G6PD, and we anticipate a similar hit rate for PfGluPho and human G6PD. This approach has the potential to generate high impact results. Malaria is the most deadly disease worldwide. The malaria parasite develops resistance to most of the currently available drugs. Thus, novel and innovative antimalarial drugs are desperately needed. PUBLIC HEALTH RELEVANCE: Tropical malaria is responsible for up to three million deaths annually. The malaria parasite Plasmodium falciparum develops resistances against most clinically available drugs. Novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase is a novel target for antimalarial drug design based on observations that humans with a deficiency in this enzyme are protected from malaria. We aim at identifying compounds that inhibit this enzyme both in malaria parasites and, to a certain extent, in humans. Our results may pave the way for the development of novel antimalarial drugs.